There is a need to quantify cellular turnover in the context of aging. We will establish the foundations of a new method to contribute crucial information to fulfill this need. This method will permit to track stable-isotope labeled cells and to measure the number of cell divisions using stable-isotopes. We will use Multi-Isotope Imaging Mass Spectrometry (MIMS), a novel methodology that we have developed, MIMS has been called "an imaging revolution" that is "beginning to yield information once considered inaccessible". We will use MIMS to image and measure stable isotope labeled DNA within nuclei of cells. This will allow us to monitor cell division with time scales potentially ranging from minutes to years, since stable isotopes do not decay, do not modify metabolism and are not toxic. MIMS has the potential to revolutionize the study of cell turnover and refreshment by permitting quantitative understanding of cellular homeostasis during aging. An emerging theory is that aging-related phenotypes might be due, at least in part, to a decline in the number or function of tissue stem cells. Tissue regenerative capacity declines with age, and in tissues such as muscle, blood, liver and brain, this decline has been attributed to a diminished number or function of tissue-specific stem and progenitor cells. Resident self-renewing cells have a significant role in the homeostatic maintenance of many organs. Numerous studies have shown that aging alters adult stem cell function. This impairment of stem cell potential could, in part, cause the progressive tissue deterioration that is observed with aging. Thus, understanding in vivo cell refreshment during aging is an important goal for understanding the aging process. Although the ability to quantify the rate of cell divisions is a crucial factor in stem cell biology, it has been extremely challenging to measure cellular refreshment in vivo. Numerous techniques exist for tracking stem cells incorporation into differentiated tissues but these techniques have important limitations. The new method that we will develop uses stable, nonradioactive isotopes to obtain highly precise quantitative data at high resolution, eliminating the need for radioactive pulses or genetic labeling. Labeling DNA with stable isotopes practically eliminates issues of toxicity and long-term dosing since there is no radioactivity and the evidence for in vivo safety of these stable isotopes is overwhelming. MIMS has sensitivities several orders of magnitude above radioactive carbon labeling and can analyze multiple isotopes simultaneously inside the same cell, permitting the quantification of incorporated isotopes from different time pulses over very long period of time and thus to describe the history of division of a single cell, which will be a boon. PUBLIC HEALTH RELEVANCE: We will develop a new methodology employing Multi-Isotope Imaging Mass Spectrometry (MIMS) to study cellular homeostasis during aging. Our tools will allow us to track stable-isotope labeled cells and measure the number of cell divisions. This method has the potential to revolutionize the study of cell turnover and regeneration and opens a powerful new way to quantify and visualize cellular homeostasis during aging.